Abrogating immune regulatory molecules such as cytotoxic T lymphocyte antigen 4 (CTLA-4) represents a new and promising strategy to induce tumor regression, stabilize disease, and prolong survival by manipulation of the immune system. An anti-CTLA-4 antibody, ipilimumab, is currently being sold for indications including melanoma. Evidence of tumor regression with prolonged time to progression has been seen in patients with melanoma who received CTLA-4 antibodies and durable responses have been observed with ipilimumab in patients with melanoma, ovarian cancer, prostate cancer, and renal cell cancer.
Full T-cell activation requires two signals. The first is initiated by T-cell receptor binding to tumor-associated antigens presented by antigen presenting cells (APCs) via major histocompatibility complexes I and II. The second signal is generated when the principal costimulatory receptor on the T cell, CD28, binds to B7 ligand subtypes CD80 and CD86 on the APC. The resulting dual signaling induces changes including T-cell proliferation and cytokine release, triggering and then amplifying the immune response. In response to T-cell activation, CTLA-4 is upregulated and competes with CD28 for CD80 and CD86 binding on APCs but with significantly higher affinity, therefore downregulating—or deactivating—the T cell (FIG. 1). CTLA-4, therefore, downregulates T-cell responses and APC function, resulting in a decreased immune response to tumor-associated antigens and immune tolerance.
The mechanisms whereby CTLA4 and PD1 exert their inhibitory effects on T-cell activation are multifaceted. CTLA4 functions primarily to limit T-cell activation and clonal expansion, whereas PD1 functions primarily to limit effector T-cell function in the peripheral tissues. Their distinct molecular structures, regulation, signaling pathways, ligand distribution, and function on Tregs and other immune cells suggest that combined therapeutic blockade of CTLA4 and PD1 could synergize to mediate anti-tumor immunity. Intlekofer & Thompson, J. Leuko. Biol. 94(1): 25-39 (2013); Hurwitz et al. Proc. Natl. Acad. Sci. USA 95: 10067-10071 (1998); Parry et al. Mol. Cell. Biol. 25(21): 9543-9553 (2005); Callahan et al. Front. Oncol. Vol 4, Art. 385 (2015).
One method by which to inhibit CTLA4-mediated downregulation is by interfering with its interaction with its ligands by binding it with a Nanobody. The possibility exists that Nanobodies, originating in llamas, could cause an unwanted anti-drug immune response, e.g., by binding of the Nanobodies by pre-existing antibodies in the patient's serum. Thus, novel methods by which to humanize Nanobodies so as to decrease or eliminate such a response are particularly valuable as are Nanobodies that are created by such methods.